CONCEPTS OF BIOLOGY

LABORATORY MANUAL

WAN NURHAYATI WAN HANAFI

 CONTENTS

Experiment 1 - Microscope- Exploring and Viewing

Experiment 2 - The Molecules of Life: Biochemistry Carbohydrates

Experiment 3 - Redox- Reaction/Catalase Activity

Experiment 4 - Specialized Cells and Staining Technique

Experiment 5 - Osmosis

Experiment 6 - Cellular Respiration

Experiment 7 - Extracellular Matrix

Experiment 8 - Mitosis and Meiosis

EXPERIMENT 1: MICROSCOPE – EXPLORING AND VIEWING

1. INTRODUCTION
After completing this lab session, you should be able to:
1. Identify the parts of compound and stereoscopic microscopes and be proficient in their
correct use in biological studies.
2. Learn the technique to measure the size of a specimen using a stage micrometer and an
ocular micrometer.

2. MATERIALS

Compound microscope, stereoscopic microscope, specimens (leaf, insects, prepared slides),

distilled water, cover slips, tissue papers, glass slides, forceps, razor blades, ocular and stage
micrometer, dropper, Petri-dishes, oil immersion

3. PROCEDURE
3a. Identify the parts of compound and stereoscopic microscopes

i. While exploring, describe your findings on the operational aspects of the microscope parts
that enables you to obtain a clear view of your samples.

ii. Next, students choose the appropriate microscope to observe samples of non-living and
living material.

iii. Make drawings and from your observation make generalities about microscope function.

iv. Describe your method to obtain field of views, levels of magnification, preparing specimen
for observation, examining the structures of samples, drawing a cell at the different
magnification levels and measuring cells.

Notes: Some terminology in microscopy

Eye-piece, objective lenses, condenser, course and fine adjustments, stage, illuminator,
diaphragm, iris, working distance, par-focal, resolution

3b. Measuring the size of a specimen using a stage micrometer and an ocular
micrometer

The magnification level of a microscope is theoretically measured by multiplying the

magnification of the ocular lens (usually 10X) by the magnification of the objective lens (4X,
10X, 40X, 100X).

In actual practice, you will find that each microscope that you use and even each lens on the
same microscope will vary slightly from the stated magnification level. Before accurate
measurements can be made, you must calibrate your microscope using both an eyepiece or
ocular micrometer and a stage micrometer. Since the ocular micrometer is inside the ocular
 lens, it will not change size when the objectives are changed. Therefore, each objective lens
must be calibrated separately.

i. The stage micrometer has calibration marks at known intervals (0.01 mm for those used in
this class.) With the lowest objective in place, focus on the stage micrometer. Rotate the
ocular micrometer until it is exactly superimposed on the stage micrometer with the
forward edge of both scales being precisely even. Then look for another point, as far from
the beginning as possible, where the two scales again overlap.

Figure 1.1: Stage and Ocular

micrometer under compound
microscope

ii. Count the spaces on each scale from the start to the second point of overlap. This will give
you X ocular units which are equal to xmm. Simply divide the number of ocular units into
the stage units or mm to determine the number of mm in each ocular unit. Most
measurements of microscopic structures are given in micrometers or microns (mm). Since
there are 1000 mm in each mm, you can multiply your mm figure by 1000 to determine the
number of mm in each ocular unit.

Remember: this calibration must be done separately for each objective lens!
3c. Introduction to immersion oil microscopy

The most powerful lens of the light microscope is

the 100x oil immersion objective. Because light is
refracted every time it passes through a medium
with a different refractive index, (air to glass or
vice versa) the quality of the image is reduced
with each passage. Thus, by reducing the number
of such passages to a minimum, the clarity,
brilliance and resolving power is preserved. Thus,
with oil immersion, there is no refraction of light
when it passes from glass to oil and vice versa.
You can see the difference between 400x and
1000x in the image to the right.

Three important rules attend the use of this lens:

1. Never use an oil immersion lens without
the oil.
2. Never get oil on any other lens.
3. Clean up all oil when finished.
Figure 1.2: Images before and after using oil
immersion.

PROTOCOL:
i. Focus at low power on a region of a smeared and stained specimen which is well-spread
and stained (not too thin, nor too thick).

ii. Rotate turret to 40x objective, locate desired portion of specimen in the center of the field.
Refocus very carefully so that the specimen is focused as sharply as possible. (Do not alter
focus for the following steps)
iii. CRITICAL STEP: Partially rotate turret so that 40x and 100x objectives straddle the
specimen. Place a small drop of oil on the slide in the center of the lighted area.
(Take care not to dribble on the stage.)

iv. Rotate turret so that the 100x oil immersion objective touches the oil and clicks into
place. Focus only with fine focus. Hopefully, the specimen will come into focus easily. Do not
change focus dramatically. If you still have trouble, move the slide slightly left and right,
looking for movement in the visual field, and focus on the object which moved.

v. With more than one specimen on a slide, do not alter focusing, rather, place a drop of oil on
the second specimen, and slide the slide laterally until it is in place.
Never go back to the 10x or 40x objectives after you have applied oil to the specimen
since oil can ruin the lower power objectives. [The 4x objective can be used because it is
high enough to be above the oil.]

Clean up! When you have finished for the day, wipe the 100x oil immersion objective
carefully with lens paper to remove all oil. Wipe oil from the slide thoroughly with a
Kimwipe. Cleanse stage should any oil have spilled on it.
4. RESULT
4a. Identify the parts of compound and stereoscopic microscopes

Figure 1: Parts and function of compound microscope

Figure 2: Parts and function of stereoscopic microscope

4b. Measuring the size of a specimen using a stage micrometer and an ocular
micrometer

Table 1: Calibrated value of ocular division (OD) for every magnification

MAGNIFICATION OBSERVATION CALIBRATION
(1OD = 1mm)
4 x 10

10 x 10

40 x 10

100 x 10
 Table 2: Calibrated value of a desired cell of prepared slide
MAGNIFICATION OBSERVATION CALIBRATION
(1OD = 1mm)
4 x 10

10 x 10

40 x 10

100 x 10
4c. Observing the specimen using a stereoscopic microscope

Table 3: Observations of ant and leaf using stereoscopic microscope

Specimens Observation Magnification
Ants

Leaf
EXPERIMENT 2: THE MOLECULES OF LIFE ̶ BIOCHEMISTRY
CARBOHYDRATES

1. INTRODUCTION

Carbohydrates serve as energy sources and provide structural support as in the cell wall of
plants. Carbon, hydrogen and oxygen are the elements found in carbohydrates.

After completing this lab topic, you should be able to:

1. Identify the reducing sugar using the Benedict’s reagent.
2. Identify the presence of starch using IKI stain.

2. MATERIALS

Dropper, Benedict’s reagent, IKI stain, Amylase solution 1%, dilute solution of sucrose, glucose,
maltose and starch, distilled water, bottle of Pepsi, potatoes, test tubes, Petri-dishes, blades.

3. PROCEDURE
2a. Benedict’s test for reducing sugar

Benedict's reagent is used as a simple test for reducing sugars. A reducing sugar is a
carbohydrate possessing either a free aldehyde or free ketone functional group as part of its
molecular structure. Recall from lectures that functional groups are the regions of a molecule
that gives it particular properties. A single molecule can have more than one functional group as
part of its structure. When a molecule with multiple functional groups is involved in a reaction
all, some or none of the functional groups may be involved.

Glucose is a reducing sugar, while the disaccharide sucrose is not. As a result, glucose heated in
Benedict's reagent reduces Cu++ ions to form a green to brick-red precipitate depending on the
amount of sugar present.

i. In the lab you prepared 5 tubes:

Tube 1: 2 ml water and 2 ml Benedict’s reagent

Tube 2: 2 ml glucose and 2 ml Benedict’s reagent
Tube 3: 2 ml sucrose and 2 ml Benedict’s reagent
Tube 4: 2 ml maltose and 2 ml Benedict’s reagent
Tube 5: 2 ml starch and 2 ml Benedict’s reagent

ii. Put all tubes in water bath at 60°C and time for the color changes.
2b. IKI test for starch

i. Cut a small section of potato and place it in a Petri-dish. Place a drop of IKI (iodine
potassium iodide) solution on the surface of the potato. IKI is a stain routinely used to
locate starch deposits. A dark blue-black color indicates that starch is present. Repeat with
starch solution.

2c. Test for sugar in Pepsi drink

Test the drink with the Benedict’s reagent and IKI stain. Remember, with Benedict’s reagent
you have to put the test solution in a hot water bath for a few minutes. You will need to time
this change. The iodine does not need heating.

i. Prepare two (2) test tubes containing 2ml Pepsi drink.

ii. Add 2ml the first test tube with Benedict’s reagent and the second test tube add 2ml IKI
stain.
iii. Add 2 ml of amylase sugar to each test tube containing the drinks and put in a water bath
at 40°C for 5-10 minutes.
iv. Observe the color changes.
v. Test tube containing Benedict’s reagent will further placed into water bath.
vi. Observe the color changes.
vii. Record your results in a table of your own design.

4. RESULT
3a. Benedict’s test for reducing sugar

Table 1: Record the color changes every 30 seconds for Benedicts test reducing sugar
Time
Test tube 1 Test tube 2 Test tube 3 Test tube 4 Test tube 5
(sec.)
30
60
90
120
150
180
210
240
270
300
330
360
390
420
450
480
510
540
3b. IKI test for starch

(a) (b)

Figure 1: The IKI test for starch of a slice of potato (a) and starch solution (b)

3c. Test for sugar in Pepsi drink

(a) (b)

Figure 2: The Benedict’s test (a) and IKI test (b) for Pepsi drink

5. QUESTIONS

4a. Benedict’s test for reducing sugar

1. Which sugar is a reducing sugar?

2. Why water is used in tube 1?

3. How long it takes for each tube to change color?

4b. IKI test for starch

1. Does the potato tissue contain starch?

2. Why would the potato contain starch?

EXPERIMENT 3: REDOX –REACTION/CATALASE ACTIVITY

1. INTRODUCTION

In this lab, you will study an enzyme that is found in the cells of many living tissues. The name of
the enzyme is catalase. It speeds up a reaction, which breaks down hydrogen peroxide, a toxic
chemical, into 2 harmless substances--water and oxygen. The reaction is as follows:
2H2O2 ----> 2H2O + O2
This reaction is important to cells because hydrogen peroxide (H2O2) is produced as a by-
product of many normal cellular reactions. If the cells did not break down the hydrogen
peroxide, they would be poisoned and die.

After completing this lab topic, you should be able to:

1. Demonstrate reduction-oxidation reaction on living tissue
2. Compare enzyme catalysis on plant and animal tissues
3. Measure the rate of enzyme reaction

2. MATERIALS

3 % hydrogen peroxide, Soaked peas, Potato, An apple, Chicken meat/liver, Razor blades
Limes juice, Test tubes and racks, Ice, Beakers, Petri-dishes, Pipettes, burette, Water bath, Lime-
juice, Test tube holder, conical flasks, weighing balance.

3. PROCEDURE
3a. Observing redox reaction

i. Cut three small slices of potato and apple and put them on a Petri-dish. Expose the first
slice to air, the second with water and the third with lime juice. Give similar treatments
for both potato and apple and observe the color change.

ii. After 30 minutes, wet the first slice of both potato and apple with lime juice and compare
the color with the first slices.

iii. Describe your findings.

3b. Comparing catalase activity in living tissues

i. Place 2 ml of hydrogen peroxide in each of 5 clean test tubes containing the following:
Tube 1: add a small piece of potato
Tube 2: add apple
Tube 3: add chicken meat
Tube 4: add liver
Tube 4: add 5 pieces of soaked peas (remove the seed coat)

ii. Grade the reaction by observing the amount of bubbles produce on a scale of 0-5 (0=no
reaction, 1=slow... 5= very fast). Feel the temperature of each test tube with your hand.
Has it gotten warmer or colder?
3c. Effect of Temperature on Catalase Activity

The rate of a reaction will be detected by measuring the amount of substrate remaining after
some period of reaction time. H2O2 is the substrate and H2O2 is converted to H2O + O2 as the
reaction proceeds. Rather than measuring the formation of product as an indicator of the
reaction, we will measure the disappearance of substrate. Incubate tubes containing Catalase
and Hydrogen Peroxide at different temperatures and examine the rate of enzyme activity by
titrating any remaining (unreacted) substrate. This will give us an indirect measure of the
substrate converted to product and therefore the rate of the reaction.

(Remaining substrate = Starting amount - amount converted to product).

2 H2O2 → 2H2O + O2

For example:

We start with 3 ml of H2O2. Add Catalase. Wait three (3) minutes. Titrate and find that 1 ml of
H2O2 remaining. Thus, how much H2O2 was consumed in the reaction?

If we had repeated the experiment, measured and found 2 ml of H2O2remaining, would this
second reaction have a higher or lower rate of reaction?

i. In the lab, you prepare 4 clean test-tubes and add 3 ml H2O2 into each.

ii. Next place each test tube to its respective temperature environment and equilibrate the
tubes for 20 minutes;

Tube 1 – 4°C
Tube 2- 24°C
Tube 3- 34°c
Tube 4- 44°C

iii. Take 10 pieces of soaked peas (dab excess water with tissue paper) and weigh. Add them
into each test tube. Let the reaction to occur for 5 mins.

iv. Then add 3.5 ml H2SO4 to each tube to stop the reaction. Pour the content of each tube
(liquid only) into a conical flask and titrate the solution with KMNO4.

v. Add KMNO4 until the solution turns pink color. This volume will show the remaining
substrate in each tube.

vi. Repeat step 1-5 with equal amount of potato. Compare catalase activity in both peas and
potato.
4. RESULT
3a. Observing redox reaction

Condition: Air Water Lime Juice

Before

After

Figure 1: Redox reaction in potato at three different treatments

Condition: Air Water Lime Juice

Before

After

Figure 2: Redox reaction in apple at three different treatments

 Condition: Potato Apple
Before
(Air)

After
(Lime
Juice)

Figure 3: Redox reaction for potato and apple after 30 minutes exposed with air and add with
lime juice

3b. Comparing catalase activity in living tissues

Table1: Catalase activity in living tissues

Grade of reaction
Condition: Observation
(formation of bubbles gas)
Test tube 1
(Potato)
Test tube 2
(Apple)
Test Tube 3
(Chicken)
Test tube 4
(Liver)
Test tube 5
(Peas)
3c. Effect of Temperature on Catalase Activity

Table2: Data titration for pees

Test Temperature Weight Initial Final Volume Description
tube reading reading used
(ͦ C) (g) (ml) (ml) (ml)

Table3: Data titration for potato

Test Temperature Weight Initial Final Volume Description
tube reading reading used
(ͦ C) (g) (ml) (ml) (ml)

4
EXPERIMENT 4: SPECIALIZED CELLS AND STAINING TECHNIQUE

1. INTRODUCTION

Cell structures fit its functions. Some cells are highly specialized in terms of structures and
functions for example, gamete cells only involved in fertilization process. The morphology of
gametes differs among species. Some cells are specialized for fixing nitrogen from the
environment. This can be observed in the photosynthetic cyanobacteria that contains heterocyst.
Not all cells are pigmented like the chloroplast. Most are translucent and hard to reveal though
the light microscope. In order to study their structures specific dyes (stains) can be used.

After completing this lab topic, you should be able to:

1. Observe and distinguish specialized cells in the prokaryotic and the eukaryotic
organisms.
2. Learn staining technique and its effect on the cell structure.

2. MATERIALS
Sterilized lancet, cleaned microscope slides and cover slips, 95% absolute ethyl or methyl alcohol,
distilled water, Giemsa stain, Coplins jars/low containers, compound microscope, stereoscopic
microscope, fresh specimens (hibiscus flower and pollen grains, Nostocs, cheek cells and blood
cells), Prepared slides (adipose cells, nerve cells, sperm cells, pollen grains, white blood cells),
distilled water, tissue papers, forceps, toothpicks, razor blades, dropper, Petri-dishes and clean
cotton.

3. PROCEDURE
3a. Observing specialized cells

i. Observe and identify all the specialized cells from fresh specimens and prepared slides and
label their parts.

3b. Staining technique

i. Cleanse a finger.
ii. With a sterile lancet, make a puncture on a fingertip and press hard.
iii. Put a drop of blood on a clean slide. Smear the blood (refer the method below) and let it dry.
Then, fix the dried smear for 10 mins in absolute alcohol (methanol/ethanol).
iv. Check the smear under the microscope.
v. Stain the slide for about 20 minutes with Giemsa stain and renewing the stain about four
times.
vi. Then rinse the slide with distilled water at a room temperature. Drain of the water and
leave the slide to dry.
vii. Observe the slide again under microscope.
viii. Repeat the technique with cheek cells.
 • Place a small drop of blood near an end of a
slide. According to figure 4.1, bring the
edge of another slide in contact with the
drop and allow the drop to bank evenly
behind the spreader. The angle between
the two slides has to be 30-40 degrees.

• Now, push to the left in a smooth, quick

motion. The smear should cover about half
the slide.

Notes:
▪ It is important that the quantity of
blood is not excessive; otherwise, the
red cells could hide the leukocytes.
So, if you succeed in making a gradual
transition from thick to thin in your
smear, you should get a zone with a
satisfactory distribution of cells.

▪ With a single drop of blood, you can

Figure 4.1: How to prepare a blood smear make several smears. In fact, to make
a smear, it is enough to leave a spot of
blood of 3 mm about in diameter on
the slide. It is useful to perform many
smears. In fact, not always they are
successful, and with some attempts, it
is easier to get one well prepared. To
avoid producing clots, you must make
each smear with fresh blood and
straight after having deposited it.

▪ To this purpose, it is useful to be

helped by another person where one
deposits the blood, and the other
makes the smears. With the
microscope, you should observe the
smears to check that some of them
are properly made. The red cells
must not overlap each other, nor be
so scarce as to be too spread out.
Figure 4.1: Making the smear
4. RESULT
4a. Observing specialized cells

Prepared slides Observation Magnification

Adipose Tissue

Human Blood Film

Lily anther
mature pollen

Mammal nerve
teased

Mixed Pollen
Grains

Nostoc

Sciatic nerve cell

Spermatozoa

Pine Germinated
Pollen

Figure 1: Prepared slides under compound microscope

 Fresh specimen Observed fresh specimen under
Magnification
(Hibiscus flower) stereoscopic microscope

Male flower parts

Female flower
parts

Figure 2: Observing fresh specimen (Hibiscus flower) under stereoscopic microscope

3b. Staining technique

Human red blood cells Buccal (cheek) cells

a. Fixing with alcohol a. Fixing with alcohol

b. Stain with Giemsa b. b. Stain with Giemsa

Figure 3: Observing Human red blood cells and Buccal (Cheek) cells under compound
microscope.
EXPERIMENT 5: OSMOSIS

1. INTRODUCTION

Osmosis is a passive transport of water across cell membrane from a hypotonic to a hypertonic
environment.

After completing this lab topic, you should be able to:

1. Describe the effects of water gain or loss in animal cell.
2. Describe the effects of water gain or loss in plant cells.

2. MATERIALS
Potato slices, Elodea leaf/onion scales, Razor blades, Digital balance/String/Ruler, Varying molar of
Sucrose solutions (0.01 M, 0.02M, 0.05M, 0.1M, 0.3M, 0.5M), Tissue papers, Test tubes, watch glass
and forceps.

You are provided with varying molar of sucrose solutions ranging from the lowest to the highest
molar (0.01M to 0.5M, respectively). Your task in this experiment is to design an experiment to
show water moves across cell membrane. Which solution contains more water? Why must you
have solutions with different molarity?

3. PROCEDURE
4a. The effect of osmosis in potato cells

1. The potato slices (3 slices /2 ml of each solution)

Things to consider:
• How many slices are adequate for the experiment?
• How big the potato slices would be? How would you prepare the potato slices? Hint: A
greater movement of water can be detected when the surface area per volume ratio of
the potato slice is big.
• How do you prepare a potato slice with big surface area to volume ratio?
• What is your control parameter?
• What variables would you measure from the potato slices? What changes would you
expect to occur to the potato slices before and after the experiment?
• How many minutes would you expose the slices to each solution (the longer the better?
• What parameter would you measure after treating the potato slices into the respective
solution?
• Tabulate the class results on whiteboard and compare.
• Identify the concentration which is hypoosmotic, isosmotic and hyperosmotic to the
potato cells.
3b. The effect of osmosis in Elodea leaves / onion scales

• You will study the leaf structure/morphology before and after adding the hypotonic
solution and the hypertonic solution. Can you detect any changes on its vacuole and the
plasma membrane?

• One feature you will notice in plant cells which is absent from animal cells is the cell wall.
This wall protects the protoplast and the membrane – plasmalemma.

4. RESULT
4a. The effect of osmosis in potato cells

Potato Slices Weight Sucrose Solution Weight

Sucrose (g) (%)
Solution Before After Difference Percentage (%)
Concentration Immersion Immersion (C) = (B) - (A) (D) = (C) x 100
(M) Initial Final (A)
(A) (B)
0.01
0.02
0.05
0.1
0.3
0.5
Table1: Size of Potato slices _____ cm

Potato Slices Weight Sucrose Solution Weight

Sucrose (g) (%)
Solution Before After Difference Percentage (%)
Concentration Immersion Immersion (C) = (B) - (A) (D) = (C) x 100
(M) Initial Final (A)
(A) (B)
0.01
0.02
0.05
0.1
0.3
0.5
Table2: Size of Potato slice: _____ cm (larger surface area)

Plot graph Percentage (%) of sucrose solution vs sucrose solutions concentration (M)
EXPERIMENT 6: CELLULAR RESPIRATION

1. INTRODUCTION

Cellular respiration is the general term which describes all metabolic reactions involved in the
formation of usable energy from the breakdown of nutrients. In some organisms, such as yeast,
fermentation occurs. In other organisms, aerobic respiration occurs.

Equation (1) shows the complete aerobic cellular respiration of glucose.

C6H12O6 + 6O2 -----> 6 CO2 + 6 H2O + 686 kilocalories of energy / mole of glucose oxidized

Equation (2) is the fermentation process

C6H12O6 2C2H5OH + 2CO2 + 2ATP

There are three ways cellular respiration could be measured.

1. Consumption of O2
2. Production of CO2
3. Release of energy during cellular respiration.

After completing this lab topic, you should be able to:

1. Observe and determine cellular respiration in yeast and onion cell.

2. MATERIALS
Beakers, pipettes, test-tubes, test-tube rack, cuvettes, tissue papers, 20% glucose, 20% lactose, 20%
galactose, distilled water, bromothymol blue (BTB), Yeast extract, methylene blue.

3. PROCEDURE
3a. Measure respiration rate in yeast using different substrate

Yeast is a facultative anaerobe that can survive in both aerobic and anaerobic environments in
order to meet its energy needs.

In this experiment, we will investigate which sugars yeast can use for cell respiration. When yeast
respires, they give off CO2.When CO2 reacts with water it forms weak carbonic acid solution. We
will use the indicator bromothymol blue (BTB) to monitor this reaction using spectrophotometer.
When the pH of the BTB solution is lowered, it turns yellow; decrease in absorption as respiration
proceeds. The spectrophotometer is set at 565nm and BTB is use as blank.

i. Set the spectrophotometer.

ii. Place 8 ml of 20% glucose, into a test-tube/beaker.
iii. Add 1 ml of BTB into the test-tube and mix.
iv. Place 2.5 ml of the mixture BTB and glucose into a cuvette. Add 0.2 ml yeast into it. Cover
gentle shake and place it into the spectrophotometer. Note the initial time.
v. Measure absorbance for every 30 seconds for the duration of 5 mins.
vi. Repeat the procedure with 20% sucrose, 20% lactose, 20% maltose and finally distilled
water.
vii. Compare the readings and rank order of sugars based on which ones the yeast can use most
effectively for respiration.

3b. Measure respiration rates at different temperatures

Respiratory indicator

i. Obtain 4 test tubes

ii. Fill each tube as indicated in the table below. Add yeast last.

Tube1 Tube 2 Tube 3 Tube 4

(Room Temperature) (100 °C) (Room Temperature) (Room Temperature)
5 ml yeast 5 ml yeast 5 ml water 5 ml yeast
1 ml glucose 1 ml glucose 1 ml glucose 1 ml water
0.2 ml methylene blue 0.2 ml methylene blue 0.2 ml methylene blue 0.2 ml methylene blue
2 ml distilled water 2 ml distilled water 2 ml distilled water 2 ml distilled

Note: for Tube 2- heat up the glucose and yeast mixture before adding methylene blue.

iii. Cover the tubes with parafilm

iv. Observe the initial color
v. Record the times taken for decolorization to occur

3c. Observing mitochondria in yeast/onion cells

i. Obtain a clean slide and place a drop of sucrose solution in the centre. Add two drops of
methylene blue and mix well with a toothpick.
ii. Place onion cells/yeast in the sucrose-stain mixture and cover with cover slip.
iii. Immediately view and search for tiny bluish oblong bodies in the cytoplasm of the cells that
immediately change into white/colorless structures.
4. RESULT

4a. Measure respiration rate in yeast using different substrate

Table1: Absorbance reading versus time of 5 substrates with yeast

ABSORBANCE READING WITH TIME

SUBSTRATE 1 minutes 2 minutes 3 minutes 4 minutes 5 minutes

30s 60s 30s 60s 30s 60s 30s 60s 30s 60s
GLUCOSE
SUCROSE
LACTOSE
MALTOSE
DISTILLED
WATER

Plot graph absorbance versus time

4b. Measure respiration rates at different temperatures

Table2: Measure respiration rates yeast at different indicator

Test Tube Temperature Observed Time taken Explanation
to decolorize
Test Tube 37 ̊C
1 Room temperature

Test Tube 100 ̊ C

2 Water bath

Test Tube 37 ̊C
3 Room temperature

Test Tube 37 ̊C
4 Room temperature
4c. Observing mitochondria in yeast and onion cells
Characteristics Yeast Onion scales
Under compound
microscope

Magnification
Figure 1: Image mitochondria in yeast and onion cells under compound microscope
EXPERIMENT 7: EXTRACELLULAR MATRIX

1. INTRODUCTION
The extracellular matrix (ECM) is a complex structural entity surrounding and supporting cells that
are found within mammalian tissue. The ECM is often referred to as the connective tissue. The ECM
is composed of 3 major classes of biomolecules:
1. Structural proteins: collagen and elastin.
2. Specialized proteins: e.g. fibrillin, fibronectin and laminin.
3. Proteoglycans: these are composed of a protein core to which is attached long chains of
repeating disaccharide units termed glycosaminoglycans (GAGs) forming extremely
complex high molecular weight components of the ECM.

Hyaline cartilage (Trachea)

Cartilage is a supportive connective tissue with a flexible rubbery matrix. It gives shape to the
external ear, the tip of the nose, and the larynx (voice box)—the most easily palpated cartilages in
the body. Hyaline cartilage is named for its clear, glassy microscopic appearance, which stems from
the usually invisible fineness of its collagen fibers.

Epithelial
Epithelial tissue is located on the very outside of an organ or organism (i.e. skin) or found lining
lumen (cavities in hollow organs (i.e. stomach lining). Epithelial tissue always has a free surface (no
contact with another cell). Epithelial tissue is also avascular (no blood vessels) and must depend on
underlying tissues for the nutrients and oxygen it needs to maintain homeostasis. For this reason,
epithelial tissue is usually very thin. Another characteristic of epithelial tissue is that it has little to
no intercellular matrix between it's cells. The cells are tightly packed together. Major functions of
epithelial tissue include protection, secretion, and absorption.

Bone
The term bone refers both to organs of the body such as the femur and mandible, composed of
multiple tissue types, and to the bone tissue, or osseous tissue, that makes up most of the mass of
bones. There are two forms of osseous tissue: (1) Spongy bone and (2) Compact (dense) bone.

Blood
Blood is a fluid connective tissue that travels through tubular vessels. Its primary function is to
transport cells and dissolved matter from place to place. Blood consists of a ground substance
called plasma and of cells and cell fragments collectively called formed elements.

After completing this lab topic, you should be able to:

1. Examine prepared slides of selected connective tissue such as loose connective, dense
regular connective tissue adipose, cartilage, ground bone, epidermal and blood that may help
the students understand and appreciate how tissue types combine to form organs

2. MATERIALS
Prepared slides of Hyaline cartilage, Simple cuboidal epithelium, Skin, Compact bone, Human blood
smear film
3. PROCEDURE
3a. Observing slides of selected connective tissue

i. Observe the prepared slides of selected connective tissues, identify and label their parts.

4. RESULT

4a. Observing slides of selected connective tissue

Observed prepared slides

Prepared slides under Magnification
compound microscope

Compact bone

Hyaline cartilage

Human blood
smear film

Skin, thin human

Simple cuboidal
epithelium

Figure 1: Prepared slide of connective tissues under compound microscope

EXPERIMENT 8: MITOSIS AND MEIOSIS

1. INTRODUCTION

Mitosis and meiosis are two forms of cell division in eukaryotic cells. Mitosis is a form of cell
division in which one cell (called the “parent cell”) divides to produce two new cells (the “daughter
cells”) that have the same number of chromosomes as the parent cell.

1. Interphase: The nondividing cells is in a stage called interphase. The nucleus may have one
or more dark-stained nucleoli and is filled with a fine network of threads, the chromatin.
During interphase, DNA replication occurs.
2. Prophase: The first sign of division occurs in prophase. There is a thickening of the
chromatin threads, which continues until it is evident that the chromatin has condensed
into chromosomes. With somewhat higher magnification you may be able to see that each
chromosomes is composed of two chromatids joined together at a centromere. As prophase
continues, the chromatids continue to shorten and thicken. In late prophase the nuclear
envelope and nucleoli are no longer visible, and the chromosome are free in the cytoplasm,
the spindle apparatus is made up of microtubules may pull the chromosomes toward the
poles of the cell where the two daughter nuclei will eventually form.
3. Metaphase: At metaphase, the chromosomes have moved to the center of the spindle. One
particular portion of each chromosome, the centomere attaches to the spindle. The
centromeres of all the chronmosomes lie at about the same level of the spindle, on a plane
called the metaphase plate. At metaphase you should be able to observe the two chromatids
of some of the chromosomes.
4. Anaphase: At the beginning of anaphase, the centromere regions of each pair of chromatid
separate and are moved by the spindle fibers towards opposite poles of the spindle,
dragging the rest of the chromatid behind them. Once the two chromatids separate, each is
called a chromosome. These daughter chromosomes continue poleward movement until
they form two compact clumps, one at each spindle pole.
5. Telophase: Telophase, the last stage of division is marked by a pronounced condensation of
chromosomes followed by the formation of a new nuclear envelope around each group of
chromosomes. The chromosomes gradually uncoil to form the fine chromatin network seen
in interphase, and the nucleoli and nuclear envelope reappear.
Cytokinesis: Occurs simultaneously with the end of telophase or after telophase. This is the
division of the cytoplasm into two cells. In plants, a new cell wall is laid down between the
daughter cells. In animal cells, the old cell will pinch off in the middle along a cleavage
furrow to form two new daughter cells.

Meiosis is a form of cell division in which each of the daughter cells of the division has one-half the
number of chromosomes as the parent cell, meiosis occurs in the cells that, ultimately, will become
the gametes (eggs or sperm) of the organism.

After completing this lab topic, you should be able to:

1. Identify and recognize the various stages of mitosis and meiosis in plant and animal cell.
2. MATERIALS
Prepared slides of anion root tip, carnoy fixative (1 glacial acetic acid: 3 methanol), compound
microscope, coverslips, eyedropper, hydrochloric acid 18%, onion set, safety glasses, slides,
toluidine blue 2%, water, forceps, watch glass, toothpicks, blades.

3. PROCEDURE
3a. Examine mitosis in onion root tips (prepared slide)

Examine the prepared slides of anion root tips. Locate the meristematic region of the onion with the
10x objective and then use the 40x objective to study the individual cells. Roots consist of different
regions. The root cap functions in protection. The apical meristem is the region that contains the
highest percentage of cells undergoing mitosis. The region of elongation is the area in which growth
occurs. The region of maturation is where root hairs develop and where cell differentiate to become
xylem, phloem and other tissues. Identify one cell which clearly represents each phase in mitosis.

3b. Examine the mitosis in onion root tip cells (fresh specimen)

Make a squash mount slide of anion root tip cells to view them in various stages of mitosis. In
plants, mitosis occurs more frequently in meristematic areas such as the root tips. To create a
squash mount slide, the living cells of the specimen are first fixed (treated with a chemical to
prevent further mitosis), then stained and finally squashed on a slide for viewing.

i. Cut off anion root tips. Place them in 18% hydrochloric acid for 4 minutes.
ii. Transfer them to carnoy fluid with chloroform (1:1) for 4 minutes.
iii. Place one of root tips on a slide and trim away all but 1-2mm of the tip.
iv. Cover the tip with 2-3 drops of 2% toluidine blue for 2 minutes.
v. Blot away the stain, add a drop of water, cover with a coverslip, and apply pressure to the
coverslip with a pencil eraser until the cells in the tip spread out in a single layer.
vi. Mount the slide on your microscope.
vii. Use the low power objective on your microscope to look for thin layers of cells and then use
the high-power objective to observe mitotic stages in individual cells.
viii. Sketch and label the cell.

3c. Examine meiosis in prepared slides

• Lily anther
• sperm/ egg cells
4. RESULT

4a. Examine mitosis in onion root tips (prepared slide)

Phase of mitosis Onion root tip cells under Magnification

compound microscope

Interphase

Prophase

Metaphase

Anaphase

Telophase

Cytokinesis

Figure 1: Phases of mitosis in onion root tips (prepared slide) under compound microscope
4b. Examine the mitosis in onion root tip cells (fresh specimen)

Phase of mitosis Onion root tip cells under Magnification

compound microscope

Interphase

Prophase

Metaphase

Anaphase

Telophase

Cytokinesis

Figure 2: Phases of mitosis in onion root tips (fresh specimen) under compound microscope
4c. Examine the meiosis in prepared slides

Images under compound

Prepared Slides Magnification
microscope
Human sperm

Lily Anther Mature

Pollen

Figure 3: Images prepared slides of meiosis under compound microscope